There is a fundamental lack of understanding in biology on the single cell level, especially in human disease biology. This gap in knowledge encompasses both how individual cells function and also how single cells combine and collaborate to construct multi-cellular creations, such as how many cancer cells associate to form a tumor. Traditionally, bioanalysis has largely been conducted through large numbers (i.e. thousands or millions) of cells being destroyed and mixed together to form a completely artificial construct: the cell lysate. Cell lysates are prepared from a large number of heterogeneous cells, either present in tissue samples or grown in culture. Bioanalysis of lysates are an amalgamated average of many heterogeneous cells subjected to artificial sample preparation, and therefore, standard bioanalytical methods have been largely unable to define single cell biology. Recently, single cell analysis (SCA) has become possible through advancements in many experimental methods. Due to its unparalleled detection sensitivity, mass spectrometry (MS) is one of the most promising analytical methods to be deployed for single cell analysis. Single-cell mass spectrometry (SCMS) and mass spectrometry imaging (MSI) are emerging techniques that could potentially revolutionize bioanalytical science in basic scientific and biomedical research. We have developed a miniaturized sampling and ionization device, the Single-probe, capable of performing single cell mass spectrometry (SCMS) in real time under ambient conditions. The Single-probe, with a sampling tip smaller than eukaryotic cells (<10 m), can be inserted into individual living cells to sample the intracellular compounds for immediate MS analysis. The Single-probe SCMS technique has produced highly sensitive detection of intracellular compounds, including cell metabolites, lipids and anticancer compounds, inside living single cells. The Single-probe has also been used successfully in MSI experiments to reconstruct the spatial distribution of species (e.g. lipids and cell metabolites) from sections of animal tissue. Our long-term research goal is to fully establish the Single- probe based techniques in SCMS and MSI for broad utilization in bioanalysis. The objective of this application is to develop the Single-probe MS technology as a first-in-class tool for quantitative SCMS (qSCMS) in cells and for MSI-based investigation of tumors. This research objective will be accomplished through completing the following three Specific Aims: 1) create quantitative single cell mass spectrometry (qSCMS) for compounds inside of single cells from cultured cell lines; 2) use the Single-probe MS to analyze single cells in in vitro tumors; and, 3) perform Single-probe MSI measurements of the in vitro and animal-derived tumors. The Single-probe MS is an innovative technology in that it provides new experimental capabilities for single cell analysis and tissue imaging. The proposed research is significant because the Single-probe MS experimental applications (i.e. qSCMS and MSI), once developed, will allow new understandings in single cell and tumor biology that are not currently possible.